Methods of producing biologically active molecules by transfer of recombinant genes into cell in culture and into live animals have been developed. For example, DNA molecules have been introduced into cultured cells by calcium phosphate precipitation or electroporations. Graham and Van der Ebb Virology 1973, 52, 456-467; Perucho et al. Cell 1980, 22, 9-17; Chu et al. Nucleic Acids Research 1987, 15, 1311-1326; and Bishop and Smith Molecular Biology Medicine 1989, 6, 283-298. DNA molecules have also been introduced into the nucleus of cells in culture by direct microinjection. Gordon et al. Proc. Natl Acad. Sci. USA 1980, 77, 7380-7384; Gordon and Ruttel Methods in Enzymology 1983, 101, 411-433; and U.S. Pat. No. 4,873,191.
However, there are two major problems of producing biologically active molecules such as protein products on a commercially viable scale via these methods. First, bacterial expression systems frequently fail to modify the proteins properly, i.e., by glycosylation, etc. Second, the subsequent isolation of gene products from the expression systems can be extremely difficult. In bacteria, yeast, and baculovirus systems the expressed proteins are most often purified from insoluble intracellular compartments. Secreted proteins in yeast require specialized protease-deficient strains coupled with appropriate vectors with secretion signals.
Retroviral vectors have also been used to introduce DNA molecules into the genome of animals. Jaenisch et al. Cell 1981, 24, 519; Soriano et al. Science 1986, 234, 1409-1413; and Stewart et al. Embo. J. 1987, 6, 383-388. Recombinant genes have been introduced into primary cultures of bone marrow, skin, fibroblasts, or hepatic or pancreatic cells and then transplanted into live animals. There has also been success in using mammary gland-specific promoters to drive the expression of foreign proteins in these secretory glands, ultimately leading to their secretion in the resultant milk. This method has been used commercially to express human growth hormone in cows and sheep. WO 94/05782. The copious volumes of milk produced by cows and sheep make this procedure attractive. However, this method suffers from several potential drawbacks: one being that the expressed protein even at relatively high levels must be purified away from a large amount of milk proteins such as caseins, immunoglobins, lactoferrins which may also entrap the desired valuable product; another being that certain protein products may be insoluble in the calcium-rich environment of milk fluid; and another being that this method requires the use of pregnant animals which are expensive and time consuming to produce.